U.S. patent number 6,068,985 [Application Number 08/325,264] was granted by the patent office on 2000-05-30 for in vitro test for helicobacter pylori.
This patent grant is currently assigned to Provalis UK Limited. Invention is credited to Robert Llewellyn Clancy, Allan Cripps, Daniel Stiel, Campbell Witt.
United States Patent |
6,068,985 |
Cripps , et al. |
May 30, 2000 |
In vitro test for Helicobacter pylori
Abstract
Contemporary infection by Helicobacter pylori in a human or
other mammal can be detected by detecting H. pylori-specific IgG
antibody in saliva, or other mucous secretion, with an antigen
preparation from H. pylori. Diagnosis depends on detection of the
antigen-antibody complex. For improved reliability, the antigen
preparation comprises H. pylori-derived components of about 265 kDa
and about 340 kDa and is substantially free of an H. pylori-derived
component of about 440 kDa. The antigen preparation may be
immobilised onto a solid support such as a nitrocellulose
strip.
Inventors: |
Cripps; Allan (Curtin,
AU), Witt; Campbell (Bicton, AU), Clancy;
Robert Llewellyn (Newcastle, AU), Stiel; Daniel
(East Lindfield, AU) |
Assignee: |
Provalis UK Limited (Deeside,
GB)
|
Family
ID: |
4149734 |
Appl.
No.: |
08/325,264 |
Filed: |
April 26, 1995 |
PCT
Filed: |
April 29, 1993 |
PCT No.: |
PCT/GB93/00894 |
371
Date: |
April 26, 1995 |
102(e)
Date: |
April 26, 1995 |
PCT
Pub. No.: |
WO93/22682 |
PCT
Pub. Date: |
November 11, 1993 |
Foreign Application Priority Data
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|
|
|
|
Apr 29, 1992 [CA] |
|
|
2067603 |
|
Current U.S.
Class: |
435/7.32;
424/234.1 |
Current CPC
Class: |
C07K
14/205 (20130101); C12N 1/04 (20130101); G01N
33/56922 (20130101); G01N 2333/205 (20130101) |
Current International
Class: |
C07K
14/205 (20060101); C07K 14/195 (20060101); C12N
1/04 (20060101); G01N 33/569 (20060101); G01N
033/554 () |
Field of
Search: |
;435/7,7.32,12 ;514/78
;424/92 ;536/23.1 ;128/760 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
6767690 |
|
Jun 1991 |
|
AU |
|
67676/90 |
|
Jun 1991 |
|
AU |
|
67676 |
|
Jun 1991 |
|
AU |
|
0329570 |
|
1989 |
|
EP |
|
89/08843 |
|
Sep 1989 |
|
WO |
|
90/03575 |
|
Apr 1990 |
|
WO |
|
Other References
S Czinn et al., "Digestive Disease Week And The 90th Annual Meeting
of The American Gastroenterological Association", Gastroenterology,
vol. 96, No. 5, Part 2, (1989). .
B. E. Dunn et al., "Two-Dimensional Gel Electrophoresis And
Immunoblotting Of Campylobacter Pylori Proteins", Infection And
Immunity, vol. 57, No. 6, pp. 1825-1833, (1989). .
Evans et al., "A Sensitive and Specific Serologic Test for
Dectection of Campylobacter pylori Infection", Gastroenterology,
96, 1989, pp. 1004-1008. .
Witt et al., "Salivary Antibodies in the Diagnosis of Helicobacter
Pylori infection", Elsevier, Sci. Publ., vol. 1, 1991, pp. 693-696.
.
Schaber et al., "Indirect Immunofluorescence Test and Enzyme-Linked
Immunosorbent Assay for Detection of Campylobacter pylori", Journ.
Clin. Microbio., vol. 27, No. 2, 1989, pp. 327-330. .
Loffeld et al., "Diagnostic Value of an Immunoassay to Detect Anti
Campylobacter Pylori Antibodies in No-Ulcer Dyspepsia", Lancet,
1989, pp. 1182-1185. .
Pateraki, E. et al, FEMS Microbiol. Immunol. vol. 64, pp. 129-136,
1990. .
Dunn, B.E. et al, Jun. 5, 1990, vol. 265, (16), pp. 9464-9469.
.
Mobley et al, International Workshop at Deerhurst, Huntsville,
Ontario, CA, Feb. 21-24, 1991. .
Casareo, S.D. et al, FEMS Microbiol. Letters, vol. 99, (No. 1, pp.
15-22, 1992. .
Ferrero, R.L. et al, Microbial Eco. Health Dis, vol. 4, pp.
121-134, 1991. .
Faulde, M. et al, Electrophosesis, vol. 14, pp, 945-951, 1993, pp.
945-951. .
O'Toole, P.W et al, J of Bacteriol, Jan. 1991, vol. 173(2), pp.
505-513. .
S. Czinn et al., Antibody Response to Campylobacter Pylori (C.
pylori)--A comparison of the Systemic and Mucosal immune System,
Gastroenterology; May 13-19, 1989, Washington D.C. .
Owens et al, J App. Bacter., 1989, vol. 66, pp. 331-337..
|
Primary Examiner: Housel; James C.
Assistant Examiner: Portner; Ginny Allen
Attorney, Agent or Firm: Foley & Lardner
Parent Case Text
This is a national stage application under 371 and claims priority
to PCT/GB93/00894, filed Apr. 29, 1993.
Claims
We claim:
1. A method for detecting contemporary infection by Helicobacter
pylori in a mammal, the method comprising contacting a mucous
secretion from the mammal with an antigen preparation from H.
pylori for a time and under conditions sufficient for an IgG
antibody in the mucous secretion specific to the antigen component
to form a complex therewith and then subjecting the complex to a
detecting means in order to detect the complex, wherein the antigen
preparation comprises H. pylori-derived components of about 265 kDa
and about 340 kDa and an H. pylori-derived component of about 440
kDa as determined by native PAGE has been removed.
2. A method as claimed in claim 1, wherein the antigen preparation
additionally comprises H. pylori-derived antigen with molecular
weights(s) of about 160 kDa and/or about 70 kDa.
3. A method as claimed in claim 2 wherein the mucous secretion is
saliva.
4. A method as claimed in claim 1 wherein the mucous secretion is
saliva.
5. A method as claimed in claim 1, wherein the antigen preparation
is bound to a solid support.
6. A method as claimed in claim 5, wherein the solid support is a
nitrocellulose strip.
7. A method as claimed in claim 1, wherein the detection means is a
second antibody, conjugated with a reporter molecule, and which
binds to H. pylori-specific antibody found in said secretion.
8. A method as claimed in claim 7, wherein the reporter molecule is
an enzyme or fluorophore.
9. A method as claimed in claim 8, wherein the reporter molecule is
an enzyme, and the enzyme is alkaline phosphatase.
10. A method as claimed in claim 1, wherein the mammal is a
human.
11. A test kit for detecting H. pylori in mucous secretion of a
mammal, said test kit comprising
(a) a solid support having an antigen preparation of H. pylori
immobilized thereon, wherein the antigen preparation comprises H.
pylori-derived components of about 265 kDa and about 340 kDa and an
H. pylori-derived component of about 440 kDa as determined by
native PAGE has been removed; and
(b) detection means which in use detect whether IgG in said mucous
secretion binds to one or more components of the antigen
preparation.
12. A kit as claimed in claim 11, wherein the antigen preparation
additionally comprises H. pylori-derived antigen with molecular
weight(s) of about 160 kDa and/or about 70 kDa.
13. A kit as claimed in claim 12, wherein the antigen is purified
by FPLC chromatography.
14. A kit as claimed in claim 11, wherein the solid support is a
nitrocellulose strip.
15. A kit as claimed in claim 11, wherein the detection means is a
second antibody, conjugated with a reporter molecule, and which
binds to H. pylori-specific antibody found in the secretion.
16. A kit as claimed in claim 15, wherein the reporter molecule is
an enzyme or fluorophore.
17. A kit as claimed in claim 16, wherein the reporter molecule is
an enzyme, and the enzyme is alkaline phosphatase.
18. A kit as claimed in claim 15, wherein the test kit is in
compartment form and comprises:
(a) a first compartment adapted to contain the solid support having
an antigen preparation of H. pyloriri immobilized thereon;
(b) a second compartment containing a second antibody conjugated
with a reporter molecule capable of producing a signal, said second
antibody being specific against IgG antibody; and
(c) when the reporter molecule is an enzyme, a third compartment
containing a substrate for said enzyme.
19. A kit as claimed in claim 18, further comprising additional
compartments adapted to receive at least one of the assay
components comprising mucous material, diluent and buffer.
20. An H. pylori antigen preparation comprising H. pylori-derived
components of about 265 kDa and about 340 kDa and an H.
pylori-derived component of about 440 kDa as determined by native
PAGE has been removed.
Description
The present invention relates generally to a method and kit which
permit the rapid in vitro detection of Helicobacter pylori
infection in mammals. More particularly, the present invention
contemplates a method for the detection of IgG antibodies against
H. pyloni in mucous secretions and thereby provides a means to
monitor contemporary infection by the microbe in mammals.
Gut infections in mammals, and in particular humans, stimulate an
immune response in mucous secretions, such as saliva, through
activation of the common mucosal immune system. This response often
initially parallels an antibody response in serum although is
generally characterised by the presence of IgA antibodies. However,
the immune response in secretion, including saliva, rapidly
diminishes following elimination of the antigen (eg bacteria or
virus) from the body. Accordingly, the presence of antibody in
mucous secretions reflects current, ie contemporary, infection. In
the case of a microbial infection, for example, antibodies in
mucous secretions, hereinafter referred to as secretious
antibodies, reflect the current status of colonisation of the
microbe, such as in the gut, and thus is a useful monitor of
contemporary infection. Serum antibody, on the other hand, persists
for some time after the microbe is eliminated from the body. A
positive serum antibody test, therefore, reflects both past and
present exposure to antigen which is less helpful to the clinician.
A positive secretious antibody test, on the other hand, indicates
present or contemporary infection by the microbe.
The present invention arose following an investigation into
Helicobacter pylori (also known as Campylobacter pylori) infection
in the gut of mammals. The diagnosis of H. pylori infection can be
made by microscopy, microbiological culture or urease detection in
gastric mucosal biopsies, urea breath test or by the presence of
specific antibodies in serum ELISAs. It might be predicted that H.
pylori infection, being an infection of the gastric mucosa, would
elicit an IgA antibody response in gastric secretion. However
during work prior to the present invention, it was discovered that
H. pylori-specific antibody in mucous secretions is of the IgG
class and not IgA as might have been expected. Little IgA antibody,
if any, is detected. Accordingly, AU-A-9067676 is directed to the
detection of IgG in mucous secretion specific to H. pylori antigen
and thereby provides a means of monitoring current, ie
contemporary, infection by that microorganism in mammals. The
corresponding academic publication is Witt et al, Frontiers in
Mucosal Immunology 1 693-696 (1991).
The presence of IgG antibodies in the saliva of Campylobacter
pylori positive patients has received some attention in the
proceedings of the Annual Meetings of the American
Gastroenterological Association. After the disclosure by Czinn et
al of the presence of such antibodies in the 1989 proceedings,
Larsen et al concluded in the May 1991 proceedings that salivary
IgG levels are a practical, non-invasive marker of therapeutic
response during a course of antibiotic therapy. In the April 1992
proceedings, Landes et al confirmed earlier observations and
observed that measurement of salivary IgG to Helicobacter pylori is
a simple, non-invasive test for detecting H. pylori positive
patients, especially in widespread or paediatric populations where
other tests are not practical.
It is clear from the above that there is widespread interest in,
and clinical potential for, a salivary IgG based diagnostic test
for H. pylori. Equally, it is clear that such a test must be
reliable as well as convenient. Invasive techniques are known (eg
from U.S. Pat. No. 4,882,271, Schaber et al, J. Clin. Microbiol.
27(2) 327-330 (1989), Loffeld et al, The Lancet, May 27, 1989,
1182-1185 and Evans et al, Gastroenterology 96 1004-1008 (1989))
and established (eg the CLOTEST.TM. kit which is available from
Delta West Ltd, Perth, Western Australia and which detects the
presence of urease in biopsy samples). A non-invasive test must
rival the reliability of invasive tests for it to become accepted
and useful.
The present invention relates to improving the reliability of
saliva-based (and other mucous-based) IgG tests for H. pylori. It
has been found that the immunological specificity of the test
method can be improved by detecting mucous IgG by using certain
antigens from H. pylori and by avoiding the use of another.
According to a first aspect of the present invention, there is
provided a method for detecting contemporary infection by
Helicobacter pylori in a mammal, the method comprising contacting a
mucous secretion from the mammal with an antigen preparation from
H. pylori for a time and under conditions sufficient for an IgG
antibody in the mucous secretion specific to the antigen component
to form a complex therewith and then subjecting the complex to a
detecting means in order to detect the complex, wherein the antigen
preparation comprises an H. pylori-derived component of about 265
kDa and is substantially free of an H. pylori-derived component of
about 440 kDa.
The present invention thus provides an effective in vitro assay for
H. pylori infection by screening for IgG antibodies in mucous
secretions.
It has been found that if the ca. 440 kDa component is present,
false positives can occur in certain patients. If the ca. 265 kDa
antigen is absent false negatives may occur. Additionally, if a ca.
340 kDa antigen derived from H. pylori is not present, false
negatives may occur in certain patients; the presence of the ca.
340 kDa antigen is therefore highly preferred. Other antigen
components from H. pylori which may, but do not have to be, present
include those with molecular weights of about 160 kDa and about 70
kDa.
An antigen component is present, for the purposes of this
invention, if it is detectable by Western blot analysis.
Conversely, it is absent if it is not detectable by this means. The
usually accepted sensitivity of Western blot analysis is in the
order of 20 .mu.g/ml.
Molecular weights of antigen components useful in the present
invention are of necessity approximate figures, because of the
limitations of current molecular weight determination procedures.
The molecular weights specifically referred to have been obtained
by a native polyacrylamide gel electrophoresis (PAGE) system sold
by Pharmacia under the trademarks PHASTGEL.TM. and PHASTSYSTEM.TM.
using the PHASTGEL gradient 8-25. This system gives a linear
relationship between a protein's migration distance and the
logarithm of its molecular weight for the molecular weight range
50,000 to 750,000 for globular proteins. Pharmacia provide a set of
high molecular weight markers in their electrophoresis calibration
kit; this marker set was used to calibrate the gels referred to in
this specification. Those skilled in the art will be aware that
slightly different results can be obtained in different hands or
even on different occasions in the same hands, and so the
approximate molecular weight figures quoted in this specification
should be read as .+-.5% or even .+-.10%. For this reason the ca.
265 kDa antigen is sometimes referred to as the 255-275 kDa
antigen.
The expression "antigen" is used in its broadest sense and includes
whole H. pylori cells or homogeneous, near homogeneous or
heterogeneous extracts from H. pylori, all of which are capable of
binding to specific antibody in a mucous secretion; providing
always that ca. 265 kDa (and preferably ca. 340 kDa) H.
pylori-derived antigens are present and a ca. 440 kDa H.
pylori-derived antigen is substantially absent. Antigen components
contemplated by the present invention include protein,
polysaccharide or lipid or any combination thereof. Preferably, the
antigen is protein, lipopolysaccharide or cell extract of H. pylori
prepared by, for example, sonication, pressure disintegration,
detergent extraction or fractionation.
One of the most effective methods of ensuring that the necessary
antigens are present, while removing many irrelevant components, is
the use of chromatography on crude extracts (for example sonicatds)
of H. pylori cells. For example, fast protein liquid chromatography
(FPLC), such as using a SUPEROSE.TM. 6 size exclusion column, is
particularly effective when applied to a crude sonicate of cells
which has been subjected to ion-exchange chromatography. Other
methods will be well within the abilities of those skilled in the
art.
Correspondingly, one of the most effective methods of ensuring that
the unwanted ca. 440 kDa antigen is absent is the use of freeze
drying on an extract of H. pylori cells, particularly after
chromatographic purification such as FPLC. Freeze drying may be
carried out in any suitable apparatus for the purpose. The actual
equipment and conditions used will depend on the scale of the
preparation. It is preferred that the freeze drying process is
carried out substantially to completion, within the limits of the
equipment used.
The invention extends to the use of naturally occurring form of
antigens and to synthetic (eg recombinant) forms and
immunologically active derivatives, analogues and relatives
thereof. It is therefore clear that an effective antigen set for
use in the present invention can either be selectively stripped
down from an H. pylori cell preparation or built up from individual
components or mixtures of components.
In the method of the invention, antibody is detected in mucous
secretion. By "mucous secretion" is meant the secretion from
mucous-secreting epithelial cells (ie mucous membrane) such as
those which line the canals, cavities and tracts that communication
with the external air, and in particular the nose, throat,
respiratory tract, eyes, genital and urinary passages and the
digestive system. In preferred embodiments, the mucous secretion is
nasal secretion, sputum or, particularly, saliva.
The saliva or other mucous secretion may be assayed undiluted or
diluted with an appropriate diluent (such as distilled water). with
increasing sensitivities, dilution may be preferred (particularly
when collection devices are used).
The antigen preparation will for convenience and preference be
bound to a solid support. Suitable solid supports include a
nitrocellulose membrane, glass or a polymer. The most commonly used
polymers for this purpose are cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene, but the invention
is not limited to them. The solid supports may be in the form of
strips, tubes, beads, discs or microplates, or any other surface
suitable for conducting an immunoassay.
Antigen components of H. pylori useful in this invention may be
either covalently or non-covalently ("passively") bound to the
solid surface. Suitable binding processes are well known in the art
and generally consist of cross-linking, covalently binding or
physically adsorbing the antigen to the solid support.
Infection is diagnosed by means of the present invention by
detecting the formation of a complex between IgG antibody in a
mucous sample and H. pylori antigens. Some form of detecting means
is therefore necessary to identify the presence (or, if required,
amount) of the antibody-antigen complex.
The detection means may be a second antibody, conjugated with a
reporter molecule, and which is specific for at least part of the
class of H. pylori-specific antibody found in the secretion; as
explained above, that antibody is IgG.
A "reporter molecule" is a molecule or group which, by its chemical
nature, has an analytically identifiable characteristic or provides
an analytically identifiable signal which allows the detection of
antigen-bound antibody. Detection may be either qualitative or
quantitative. The most commonly used reporter molecules in this
type of assay are either enzymes, fluorophores or radionuclide
containing molecules (ie radioisotopes). In the case of an enzyme
immunoassay, an enzyme is conjugated to the second antibody,
generally by means of glutaraldehyde or periodate. As will be
readily recognised, however, a wide variety of different
conjugation techniques exist, which are readily available to those
skilled in the art. Commonly used enzymes include horseradish
peroxidase, glucose oxidase, .beta.-galactosidase and alkaline
phosphatase, among others. The substrates to be used with the
specific enzymes are generally chosen for the production, upon
hydrolysis by the corresponding enzyme, of a detectable colour
change. Substrates can be soluble or insoluble, depending upon the
chosen application. For example, 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium is suitable for use with alkaline
phosphatase conjugates; for peroxidase conjugates,
1,2-phenylenediamine-5-aminosalicylic acid,
3,3,5,5-tetramethylbenzidine, tolidine or dianisidine are commonly
used. It is also possible to employ fluorogenic substrates, which
yield a fluorescent product, rather than the chromogenic substrates
noted above. Examples of fluorogenic substrates are fluorescein and
rhodamine. When activated by illumination with light of a
particular wavelength, the fluorochrome-labelled antibody absorbs
the light energy, inducing a state of excitability in the molecule,
followed by emission of the light at a characteristic colour which
is usually visually detectable with a light microscope.
Immunofluorescence and EIA techniques are both well established in
the art and are particularly preferred for the present method.
However, other reporter molecules, such as radioisotope,
chemiluminescent, and bioluminescent molecules and/or dyes and
other chromogenic substances, may also be employed.
The choice of a particular reporter molecule conjugated antibody
will be, for the most part, determined by the intended use and user
of the present invention. Additionally, although the test is
appropriate for all mammals susceptible to H. pylori infections, it
is most applicable and useful to monitoring H. pylori infection in
humans.
Accordingly, in a preferred embodiment, the present invention
provides a method for detecting contemporary infection by H. pylori
in a human comprising:
(a) contacting a mucous secretion from the human with an antigen
preparation of H. pylori immobilised onto a solid support for a
time and under conditions sufficient for IgG antibody in the mucous
secretion specific to antigen in the antigen preparation to form a
complex therewith, wherein the antigen preparation comprises H.
pylori-derived components of about 265 kDa and about 340 kDa and is
substantially free of an H. pylori-derived component of about 440
kDa;
(b) contacting the complex with an effective amount of a second
antibody labelled with a reporter molecule and specific to the H.
pylori-specific IgG antibody; and
(c) detecting binding of the second antibody to said IgG antibody
by the reporter molecule.
Hence, a medical practitioner, clinician, nurse or even patient may
use a nitrocellulose or other suitable solid phase support membrane
strip carrying immobilised H. pylori antigens, such as freeze
dried, FPLC-fractionated soluble sonicate. The strip is then
contacted with the mucous secretion for a time and under conditions
sufficient to allow potential H. pylori specific antibodies of the
IgG class in saliva to bind to the immobilised antigens. Instead of
saliva, the source of mucous secretion may be nasal secretion or
sputum.
The test strip, once exposed to mucous secretion, is then allowed
to come
into contact with a second antibody conjugated with a reporter
molecule for a time and under conditions sufficient for the second
antibody to bind to the first antibody. Preferably, the reporter
molecule is an enzyme, such as alkaline phosphatase. The test strip
is then washed and a substrate for the reporter molecule, (in the
case where the reporter molecule is alkaline phosphatase,
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium), is
brought into contact with the strip. The substrate reacts with the
reporter molecule, thereby giving a detectable change. For example,
alkaline phosphatase hydrolyses 5-bromo-4-chloro-3-indolyl
phosphate to a purple product. This entire procedure can occur in
the medical practitioner's surgery or office.
When a more quantitative enzyme-linked immunosorbent assay (ELISA)
is required, such as in a clinical laboratory, a microtitre tray
may be used carrying immobilised H. pylori antigens in the wells of
the tray. In this case, samples of mucous secretion, eg saliva, are
added to the well to allow potential H. pylori-specific IgG
antibodies to bind to the immobilised antigen. Excess secretion is
washed away and a second antibody specific to IgG conjugated to a
reporter molecule is added to allow an antigen-antibody-conjugated
antibody complex to form. This complex is detected by adding a
substrate to the reporter molecule as described above to allow, for
example, a visual signal which may then be quantitated
spectrophotometrically or by other means.
The invention may be embodied, alternatively or additionally in an
immunoblot system, which may also be quantitative (for example by
measuring intensity of colour of the blot and/or distance migrated
through a chromatographic strip.
The present invention, especially when in the form of the
nitrocellulose strip, has many advantages over the present
commercially available assays for H. pylori. The use of mucous
secretion, and in particular saliva, to assay for H. pylori
antibody enables diagnosis of current or contemporary infection and
thus enables the medical practitioner to:
a) Link gut symptoms with H. pylori which would enable decisions to
be made with respect to further investigation (including invasive
procedures) and/or management (eg use of specific anti-H. pylori
agents). The latter may be expected to have special significance
with respect to H. pylori-associated non-ulcer dyspepsia,
gastritis, duodenal ulceration, gastric ulceration and related and
other conditions.
b) Have for the first time a convenient non-invasive test in the
doctor's room, clinic or hospital to follow patients with proven
peptic ulcer to detect early recurrence. A positive test enables
early diagnosis and prevention or early treatment of recurrent
peptic ulcer. A negative test has a reciprocal usefulness in
analysis of the diagnostic approach to dyspepsia.
Additionally, the test contemplated by the present invention
provides a simple yes/no answer, not requiring, for example, the
taking of blood. It can be read in minutes and developed without
any special preparation of specimen by the clinician. A significant
advantage of the present invention is the use of mucous secretion
(eg saliva) to test for antibodies specific to H. pylori.
According to a second aspect of the present invention, there is
provided a test kit for detecting H. pylori in mucous secretion of
a mammal, said test kit comprising
(a) a solid support having an antigen preparation of H. pylori
immobilised thereon, wherein the antigen preparation comprises H.
pylori-derived components of about 265 kDa and about 340 kDa and is
substantially free of an H. pylori-derived component of about 440
kDa; and
(b) detection means which in use detect whether IgG in the said
mucous secretion binds to one or more components of the antigen
preparation.
The detection means may be an antibody conjugated with a reporter
molecule which is capable of producing or giving rise to a
detectable signal; the antibody would be specific against IgG
antibody of the mammal in question. In a preferred embodiment, the
reporter molecule is an enzyme capable of catalysing a reaction in
which the product(s) is or are detectably different from the
reactants). In such a case, a substrate for the enzyme, which may
for example have different calorimetric properties from the product
of the enzyme, will usually also be present in the kit.
Hence, in a preferred embodiment, the present invention extends to
a kit for detecting H. pylori-specific IgG antibody in mucous
secretions in a mammal, such as a human, the test kit being in
compartment form and comprising:
(a) a first compartment adapted to contain a solid support having
an antigen preparation of H. pylori immobilised thereon, wherein
the antigen preparation comprises H. pylori-derived components of
about 265 kDa and about 340 kDa and is substantially free of an H.
pylori-derived component of about 440 kDa;
(b) a second compartment containing an antibody conjugated with a
reporter molecule capable of producing a signal, the antibody being
specific against IgG antibody; and
(c) when the reporter molecule is an enzyme, a third compartment
containing a substrate for said enzyme.
The kit may also contain additional compartments such as to receive
suitable mucous material and/or for one or more diluents and/or
buffers. The kit may also be packaged for sale in a suitable
form.
The combination of the ca. 265 kDa and ca. 340 kDa antigens in the
absence of the ca. 440 kDa antigen from H. pylori is useful in
practising the method of the first aspect of the invention and in
the preparation of kits in accordance with the second aspect of the
invention. According to a third aspect of the invention, therefore,
there is provided an H. pylori antigen preparation comprising H.
pylori-derived components of about 265 kDa and about 340 kDa and
being substantially free of an H. pylori-derived component of about
440 kDa.
Such an antigen preparation can be prepared, as previously
indicated, by freeze-drying an extract of H. pylori cells to remove
the ca. 440 kDa antigen. According to a fourth aspect of the
invention, therefore, there is provided a process for the
preparation of an antigen preparation as described above, the
process comprising freeze-drying an extract of H. pylori cells. The
extract which is subjected to freeze drying is preferably a ca. 67
kDa to ca. 440 kDa fraction obtainable by size exclusion
chromatography of a crude H. pylori extract.
Other preferred features of each aspect of the invention are as for
the other aspects, mutatis mutandis.
The following examples illustrate the invention but do not limit
its scope.
EXAMPLE 1
In this example, a crude sonicate of H. pylori is prepared,
subjected to FPLC fractionation and freeze-dried. Western blot
analysis shows that approximately 265 and 340 kDa proteins are
responsible factors in the immunological specificity of the assay
of the present invention. These proteins only show reactivity in
true positives unlike a potentially contaminating 440 kDa protein.
This protein is removed by freeze-drying, to which it appears to be
sensitive.
Preparation of Helicobacter pylori antigen
Centrifuged Sonicate, including Anion Exchange Chromatocraphy
i) Cultures of H. pylori are harvested from chocolate agar plates
in PBS. The bacteria are grown as two separate cultures, a wild
strain designated "Traub" and an NCTC strain 11637.
ii) The bacteria are washed three times in PBS by centrifugation
for 5 minutes at 10,000.times.g.
iii) Washed bacteria are resuspended in 2 ml of PBS and CFUs are
estimated by reading on a spectrophotometer at 405 nm.
iv) The suspension is subjected to 5 cycles of sonication (30
seconds at 6 .mu., followed by 60 seconds rest which constitutes
one cycle).
v) Sonicated organisms are centrifuged for 10 minutes at
10,000.times.g.
vi) The supernatant is recovered and filtered through a 0.45 .mu.m
filter, then a 0.22 .mu.m filter, to remove any remaining cellular
debris.
vii) After filtering, a protein estimation is performed. Note: If
it is not possible to process the sonicate directly, it may be
frozen down at -70.degree. C. at this point in the method until an
opportunity arises to continue the method.
viii) The MONO Q.TM. anion exchange column can take 20-50 mg of
protein per 500 .mu.l injection (so the sonicate can have up to
40-100 mg of protein per ml).
ix) At this point the sonicate usually needs concentration in order
to reduce the number of injections necessary to process the whole
sonicate, and to increase the protein concentration to the
allowable level. Concentrating is carried out using CENTRISART.TM.
concentration tubes, with spins at 2500 rpm for 20 minutes each, at
4.degree. C. The sonicate is usually concentrated in order to give
2 to 3.times.500 .mu.l injections.
x) After concentration, the preparation is put down the MONO Q
column in 500 .mu.l injections. A readout is given for each
injection and all parameters are recorded.
xi) Once the preparation has been run through the MONO Q fractions
are checked for a urease peak.
xii) The values obtained from the urease test are plotted onto the
protein profile obtained from the FPLC (see below). The required
fractions are then pooled and a protein estimation performed. This
protein concentration is used to determine the number of spins
needed to concentrate the pool to approximately 1 ml. The
preparation is concentrated using CENTRISART concentration
tubes.
FPLC Antigenic Fractions
i) Sonicate is further purified by fast protein liquid
chromatography on a SUPEROSE.TM. 6 size exclusion column.
ii) The column is equilibrated with PBS pH 7.6-7.7 containing 0.02%
w/v sodium azide.
iii) 200 .mu.l of sonicate are loaded onto the column and 0.5 ml
fractions collected.
iv) Fractions are collected and a urease test performed to find the
urease peak. The values are plotted onto the readout obtained from
the FPLC. The required fractions are pooled.
v) A small aliquot is taken to perform a native PAGE analysis to
check consistency of bonding. These fractions do not include the
urease peak but correspond to a peak representing a group of
smaller proteins with molecular weights between 440 kDa and 67
kDa.
vi) The remainder is stored at -20.degree. C. and later pooled with
other, similar preparations to form batches.
Freeze-Drying
Antigen from the above batches is freeze dried in an FSE Freeze
Drier comprising:
(a) a Leybold (USA) TRIVAC.TM. pump, model No D4A;
(b) a General Electric (USA) 240 volt AC motor;
(c) a VIRTIS.TM. vacuum chamber (Virtis Co. Inc., New York, USA);
and
(d) a refrigeration unit incorporated into the cabinet of the
freeze drier.
30 ml of antigen-containing liquid, frozen at an angle in a 5 cm
diameter 50 ml specimen jar to increase surface area, was placed
within the vacuum chamber, and the system operated. The temperature
of the system in use was always set on -55.degree. C. The vacuum of
the system in use was kept at 80-100 mTorr (approximately 0.001
atmospheres); the range value depends on the initial volume of
frozen liquid and its surface area and, hence, the amount of vapour
in the chamber. The sample was freeze-dried to complete dryness,
which took 16-18 hours.
Native Gradient-PAGE using Pharmacia's PHASTSYSTEM.TM.
Antigens from the above fractions, before and after freeze drying,
are subjected to native PAGE using linear gradient acrylamide gels
and the protein profiles visualised with rapid silver staining, as
follows.
i) Buffer strips were PHASTGEL.TM. native buffer strips to separate
proteins in their native state.
ii) The gel was PHASTGEL.TM. Gradient 8-25. This system gives a
linear relationship between a protein's migration distance and the
log of its molecular weight for the molecular weight range 50 kDa
to 750 kDa for globular proteins.
iii) Markers used were Pharmacia's electrophoresis calibration kit
for high molecular weight determinations. 1 vial (Cat. No.
17-0445-01) of lyophilised marker was dissolved in 100 .mu.l
distilled water. Reconstituted marker was diluted 1/10 for silver
staining of gel. 1 .mu.l marker was run in duplicate.
iv) For the antigen sample, 1 .mu.l of each SUPEROSE.TM. 6
FPLC-purified extract was run in duplicate.
v) Separation conditions: samples were run using optimized method
of native PAGE from PHASTSYSTEM.TM. Separation Technique File No.
120. The length of the run was 120 volt.hours.
vi) Development conditions: gel is silver stained following the
method from Table 3 from PHASTSYSTEM.TM. Development Technique File
No. 210 ("Silver Staining Method Optimised for Native-PAGE").
vii) The molecular weight determination is obtained using the high
molecular weight markers that are run with the antigens on the gel.
Standard curves are determined by plotting the log molecular weight
against the R.sub.f value.
Comparison of Antigens before and after Freeze-Drying
Using the above native-PAGE methodology, the following results were
obtained. Values are given as means plus or minus one standard
deviation. (n) indicates the number of determinations that could be
made from the gel.
______________________________________ Purified Freeze-dried
______________________________________ Antigen 1 436 .+-. 20.6 (4)
Not detected (4) Antigen 2 338.6 .+-. 14.1 (4) 334.0 .+-. 18.5 (4)
Antigen 3 248.0 .+-. 18.1 (4) 262.0 .+-. 0 (2) Antigen 4 162.7 .+-.
23.0 (3) 162.0 (1) Antigen 5 70.0 .+-. 0 (3) 70.0 (1)
______________________________________
Further Characterisation of Antigens
i) Relative molecular masses may be estimated from standard
containing thyroglobulin, ferritin, catalase, lactate dehydrogenase
and albumin.
ii) Western blotting methods may be employed to detect the
appropriate protein bands by using sera positive to H. pylori.
iii) FPLC fractionation removes those lower molecular weight bands
which are reactive with negative sera.
iv) The effect of freeze drying on the antigen preparation appears
to diminish the presence of a large molecular weight (about 440
kDa) unit which is reactive with false positive sera. The absence
of the 440 kDa band is important in the discrimination between
positive, false positive and negative sera.
The effect of this purification procedure is the identification of
a protein in the molecular weight range 255-275 kDa but generally
about 265 kDa and a protein whose molecular weight is about 340 kDa
which are reactive to positive sera but not negative sera.
EXAMPLE 2
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
1. Coating of ELISA Plates with Antigen
i) Polystyrene ELISA plates are used (Polysorb, Nunc, Denmark).
ii) Freeze dried antigen preparation from Example 1 is optimally
diluted (highest dilution giving maximum sensitivity for antibody
positive saliva without increasing reactivity of antibody negative
saliva) in coating buffer (see 2(iii) below).
iii) An aliquot of 100 .mu.l of diluted antigen is added to
"antigen" wells of an ELISA plate and 100 .mu.l of coating buffer
(without antigen) is added to "buffer" wells.
iv) Plates are incubated overnight at 4.degree. C. temperature.
v) Incubated plates are emptied, an aliquot of 100 .mu.l of 5% w/v
dried skim milk powder/coating buffer (see 2(iii) below) added for
30 minutes and emptied immediately by flicking the contents
out.
2. Buffers
i) Phosphate buffered saline (PBS): 0.14M NaCl, 0.003M Na.sub.2
HPO.sub.4, 0.001N NaH.sub.2 PO.sub.4.2H.sub.2 O in 1 liter of
deionized water adjusted to pH 7.2.
ii) Substrate buffer: 10.1 g citric acid, 14.2 g disodium hydrogen
orthophosphate (Na.sub.2 HPO.sub.4), 150 .mu.l H.sub.2 O.sub.2 (30%
w/v) in 1 liter of deionized water adjusted to pH 5.0.
iii) Coating buffer: 2.42 g TRIS[tris(hydroxymethyl) amino
methane], 58.44 g NaCl, in 1 liter of deionized water adjusted to
pH 7.5.
3. Treatment of Plates after Antigen Coating
For long term storage (6 months) of antigen coated plates, plates
are dried after coating and blocking and stored at 4.degree. C.
with a desiccant. This procedure is necessary for long term
preservation of the antigen coated plates.
4. ELISA Procedure
a) Horseradish peroxidase method
i) An aliquot of 100 .mu.l of saliva diluted 1/2 in 0.05% (w/v)
dried skim milk powder/PBST (PBSTM) or 100 ul of saliva diluted 1/2
in PBSTM is added to an antigen well and to a buffer well of the
ELISA plate.
ii) Plates are incubated for up to 90 minutes at ambient
temperature.
iii) Plates are washed 5 times by immersion in PBST.
iv) An aliquot of 100 .mu.l of horseradish peroxidase anti-human
IgG diluted optimally (highest dilution giving maximum sensitivity
for antibody positive saliva without increasing reactivity of
antibody negative saliva) in PBSTM is added to antigen and buffer
wells.
v) Plates are incubated for up to 90 minutes at ambient
temperature.
vi) Plates are washed 5 times by immersion in PBST.
vii) An aliquot of 100 .mu.l of horseradish peroxidase substrate
(Product T-2885, Sigma, USA) in substrate buffer is added to
antigen and buffer wells.
viii) Plates are incubated for up to 30 minutes at ambient
temperature.
ix) 100 .mu.l of 1M H.sub.2 SO.sub.4 is added to antigen and buffer
wells.
x) For each serum or saliva sample, the absorbence (A) of the
buffer well is subtracted from the A of the antigen well and the
resultant A is converted to ELISA units using a standard curve
(constructed from doubling dilutions of a standard antibody
positive serum).
xii) A survey of 100 saliva from patients who had been demonstrated
by biopsy to be infected (or not infected) is used to determine the
number of ELISA units corresponding to infection.
EXAMPLE 3
IMMUNOBLOTTING ASSAY
1. Preparation of Helicobacter pylori Antigen
The procedure for the preparation of H. pylori antigen for the
immunoblotting assay is identical to that in Example 1.
2. Coating of the Membrane with Antigen
i) Nitrocellulose membrane is used. Nylon based membranes can also
be used.
ii) After blotting dry, the membrane is soaked for 5 minutes in an
optimal dilution of antigen (highest dilution of antigen giving
maximum sensitivity with antibody positive samples without
producing positive reactions in antibody negative samples).
iii) The membrane is then incubated for 30 minutes in 5% (w/v) skim
milk powder/TBS.
iv) The membrane is washed twice for 5 minutes in 0.05% (w/v)
polyoxyethylene sorbitan monolaurate/TBS (TBST). For long term
storage (up to 18 months) the membrane is then dried and stored at
4.degree. C. with a desiccant.
v) The membrane is immersed for up to 5 minutes in undiluted test
saliva in a test bottle or under the tongue.
vi) The membrane is washed for 30 seconds under running tap
water.
vii) The membrane is immersed in alkaline phosphatase-conjugated
anti-human IgG optimally diluted (to enable distinction between
antibody positive and antibody negative saliva) in PBSTM for up to
5 minutes.
viii) The membrane is washed for 30 seconds under running tap
water.
ix) The membrane is immersed for 5 minutes in substrate (0.3 mg
nitroblue tetrazolium, 0.15 mg 5-bromo-4-chloro-3-indolyl phosphate
in 1 ml 0.1 M NaHCO.sub.3, 1.0 mM MgCl.sub.2, pH 9.8).
x) Antibody positive samples produce a mauve colour change in the
antigen region of the membrane whereas antibody negative samples do
not alter the colour of the membrane in the antigen region.
EXAMPLE 4
Patients referred to the Gastroenterology Department, Royal North
Shore Hospital, NSW, for investigation of upper gastrointestinal
symptoms were studied. Microbiological culture, microscopy for the
detection of H. pylori, urease tests, and histology of gastric
biopsies and ELISAs were performed on all patients where
possible.
Correlation of Salivary Antibodies with Biopsy Tests for H.
pylori
The relationship between gastric biopsy histology and saliva ELISA
results together with other tests for H. pylori infection are shown
in the following table.
A serum ELISA has been shown to be more sensitive and specific than
the biopsy tests in detecting H. pylori infection and is becoming
generally accepted as the "gold" standard for detection of H.
pylori infections. The results in this table show that the salivary
assay system performs equally as well as serum. Where no
inflammation or chronic inflammation is present, all tests
currently available, including urease, culture and histology,
perform relatively equally. However, patients with active
inflammation are indicated as having H. pylori infection more often
with both the serum and salivary ELISA than with other systems. The
reason for this may be the fact that the biopsy is a fairly random
sampling procedure and may miss areas of H. pylori infestation. As
can be seen the saliva ELISA results correlate well with the serum
ELISA results.
______________________________________ RELATIONSHIP BETWEEN ELISAs
AND BIOPSY TESTS WITH GASTRIC HISTOLOGY Gastric Serum Saliva CLO
Culture Histology Inflammation - + - + - + - + - +
______________________________________ NONE 5 0 5 0 5 0 5 0 5 0
CHRONIC 3 1 3 1 3 1 3 1 3 1 ACTIVE 1 12 2 11 4 8 4 8 3 10
______________________________________
Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
* * * * *